We propose to investigate the mechanism(s) that regulate a cell's ability to escape from the crisis caused by gradual telomere shortening. In particular, we will define the roles that the i) A-NHEJ (alternative-non- homologous end joining) pathway of DNA DSB (double-strand break) repair as well as the ii) chromatin remodeling gene, ATRX (alpha thalassemia/mental retardation syndrome, X-linked), play in this process. As normal human cells age, their telomeres gradually shorten. When the telomeres shorten significantly, the cell undergoes senescence, which is a naturally-occurring, non-proliferative barrier to cancer. If, however, a cell should suffer a transforming mutation, it can by-pass senescence and continue to proliferate until its telomeres become so short that they are non-functional. The resulting lack of end protection triggers crisis, a state that is highlighted by genomic instability as chromosomes engage in breakage:fusion:bridging cycles that almost invariably result in the death of the cell. On rare occasions a cell can reestablish its telomeres and stabilize its genome. Such cells are said to be immortalized and it is likely that they are the progenitors of most human cancers. That the (dys)regulation of telomere maintenance is also associated with aging, immortalization, and tumorigenesis in other experimental systems adds confidence to the belief that these issues are conserved and important. Here, we demonstrate that the genes LIGIII (DNA ligase III) and PARP1 {poly(ADP) ribose polymerase 1} are required for human cells to survive the crisis induced by gradual telomere shortening. LIGIII and PARP1 function in the A-NHEJ branch of DNA DSB repair. We hypothesize that it is the absence of A- NHEJ that results in the death of cells undergoing crisis and we propose to i) use structure:function approaches to define the molecular interactions required for the process, ii) identify other genes involved in crisis survival using directed approaches and genome-wide screens and iii) begin to test models for how LIGIII might mechanistically control this process. In addition, we describe our preliminary data demonstrating that ATRX is a crucial regulator of ALT (alternative lengthening of telomeres) and we describe an experimental system in which we can study the genesis of ALT. In all of these approaches we utilize the strengths of the Hendrickson and Baird laboratories. The Hendrickson laboratory excels at the technology of gene targeting to study the impact of loss-of-function mutations of genes (LIGIII, PARP1 and ATRX in this instance) on telomere maintenance. The use of gene targeting provides a facile experimental system in which null, hypomorphic, and/or conditional mutations can be introduced with rapidity into human somatic cells. The Baird laboratory is the world's leader in analyzing telomere fusion events in human cells undergoing crisis. Their ability to characterize the dynamics of single telomeric ends has provided the field's deepest understanding of the mechanism of telomere fusions in human cells. In summary, our proposed studies impact on DNA repair and telomere maintenance and the importance of understanding these processes for cancer biology is clear.